Antifolate-sensitive Chinese hamster lung (CHL) cells have been shown to synthesize two molecular weight classes of dihydrofolate reductase (DHFR). Each class contains two isoelectric forms of the protein and all four DHFRs have been shown to be encoded by mRNA. Although the number of genes responsible for the synthesis of these different DHFRs is not clear, a variety of experiments has shown that the parental cell line DC-3F is heterozygous at the DHFR locus. When challenged with antifolate drugs, one or the other allele is amplified, thus accounting for the over-production of one or the other of the two molecular weight classes of DHFR and their associated isoelectric forms. Additionally, these alleles display different Southern patterns, and Northern blots show that their multiple DHFR mRNA transcripts are present in substantially different abundancies. Additionally, it has been shown that, in the absence of selection pressure, the DHFR gene amplification is unstable in these cells, and that the chromosomally localized amplification units (HSRs) are lost at the rate of about 150 kb of DNA per cell division. With the use of recombinant DNA technology, DNA transfection techniques, restriction endonuclease mapping, and DNA sequencing techniques, we propose: (1)\to define the gene complement of the DHFR locus and determine the nature of its heterozygosity, as well as the molecular basis for the isoelectric forms of DHFR within each molecular weight class; (2)\to characterize those structural features of the different genes that are responsible for the observed abundant differences in their multiple DHFR mRNAs; and (3)\to attempt to isolate from reverting cell lines extrachromosomal copies of the DHFR gene and its flanking regions. Characterization of these extrachromosomal sequences may shed new light on the process of deamplification, i.e., reversion, at a time when the overall regulatory implications of the amplification phenomenon are being reevaluated in the context of malignant transformation and differentiation functions. (I)